PROJECT SUMMARY Corticotropin Releasing Factor (CRF) is a neuropeptide that is partly responsible for generating the physiological symptoms of stress, and can induce fear and related anxiety behaviors. CRF expression is particularly enriched in brain regions that are responsible for regulating threat-adaptive behaviors, such as the central amygdala (CeA). In addition, the CeA is found to be hyperactive in several stress related neuropsychiatric disorders. The direct relationship between increased CRF signaling and the production of fear and anxiety phenotypes is emphasized by clinical findings that patients suffering from PTSD have elevated cerebrospinal fluid levels of CRF. However, whether amygdalar neurons that contain CRF (CRF+ neurons) are able to mediate the expression of fear and anxiety behaviors remains a mystery. Does the activity of CRF+ neurons in the central amygdala correspond with the expression of fear and anxiety related behaviors? Do these neurons form a nexus with other brain regions that are recruited during threat exposure? Can decreasing the activity of CRF+ neurons serve as a novel targeted therapy for treating neuropsychiatric anxiety disorders? In order to address these questions, a rigorous training plan involving molecular, systems, and behavioral neuroscience techniques will be employed. Electrophysiological, optogenetic, pharmacogenetic, and behavioral experiments will be performed to test the hypothesis that CRF+ neurons of the CeA are positioned to form strong anatomical connectivity with brain regions that are recruited during fear and anxiety expression, and that increased activity of these neurons directly contributes to the behavioral manifestations of stress induced anxiety. We will probe the connectivity of CRF+ neurons in the CeA with upstream and downstream brain regions known to form projections to and from the CeA, respectively. ChR2 will be virally expressed in excitatory brain regions that send visceral, autonomic, and endocrine afferent information to the CeA and the relative input strength from these regions onto neighboring CRF+ and CRF- neurons will be compared using whole-cell patch clamp electrophysiology during light stimulation. We hypothesize that excitatory brain regions that project to the CeA, which are recruited during behavioral expression of fear and anxiety behaviors, will provide greater input strength to CRF+ neurons. Furthermore, we predict that CeA CRF+ neurons send output projections to brainstem nuclei that are involved with generating specific threat-adaptive behaviors characteristic of fear and anxiety expression. Lastly, we will use viral expression of designer receptors exclusively activated by designer drugs (DREADDs) as a proof-of-principle approach to test whether increases or decreases in CRF+ neuronal activity in the CeA corresponds with increases or decreases in fear and anxiety behaviors. Completion of these studies could validate cell-type specific and brain region specific targeting of CRF+ neuronal activity as a novel therapeutic approach for treating stress induced anxiety symptoms found in many neuropsychiatric mood disorders.